Traditional pharmaceutical process technology for manipulating the physical properties and water solubility of these important anti-cancer drug moieties has been to render them water soluble with strong mineral acids. Salt formation is reserved as the final step in the synthesis, as described by Brana and associates (U.S. Pat. No. 5,420,137; 1995) more as an after thought for formulation purposes than as an integral or even strategic component, of the reaction synthesis. Such mono and divalent mineral acid salts retain hygroscopicity, and the divalent species, which form hydrates, have also been found to be incompatible with many pharmaceutical auxiliaries required for preparing sterile injectables, tablets or gelatin capsules.
In contradistinction to the prior art and accepted practice, we have found that the early incorporation of organic acids into the synthetic elaboration of amonafide and its aminoalkyl analogs moieties permits more rapid isolation and purification of intermediates, higher concentrations of reactants during the synthetic process, and more desirable properties, such as bulk density, flocculence and compressibility.
Furthermore, the resulting organic salts show higher solubilities in water as well as in osmotically balanced electrolyte solutions, which otherwise would be incompatible via common ion effects with inorganic, mineral acid salts such as the hydrochlorides and methylsulfonates used routinely heretofore. Moreover, organic acid salts of the present invention retain a higher degree of amphiphilic compatibility both with protic and aprotic solvents of varying polarity, thereby affording a broader range of crystallizing conditions for purposes of purification and isolation than would be afforded by the corresponding mineral acid salts.
Examination of the process chemistry for amonafide readily illustrates the shortcomings of the prior art and the adverse properties of the resulting mineral salts, which have been circumvented by the present invention. Among the synthetic approaches described in the patent and professional literature, the common denominator requires acylation of 1-amino-2,2-N,N-dimethylamino ethylene diamine, or its similarly substituted homologs, with a polycyclic, substituted aryl anhydride as shown in FIG. 1. Thus for amonafide, in accordance with the method of Brana and Sanz (Eur. J. Med. Chem 16:207, 198.1) compounds I and II in FIG. 1 are combined in ethanol to afford a precipitate of mitonafide, which must then be recrystallized multiple times from a larger volume of ethanol to be freed of tarry-black or brown by-products.
While the acylation may be conducted at a concentration of 1 gram of precursor anhydride in 25 ml of solvent, recrystallization of mitonafide requires three recrystallizations at a concentration of 1 gr in 75 ml to afford light cream colored material, free of tarry substances and exhibiting a constant melting. Although the initial yields according this process range within 60-80 percent, subsequent purification reduces the net yield to 30% of material with sufficient purity for subsequent conversion into a pharmaceutically acceptable end-product.
These isolation and purification conditions also apply to the synthesis of mitonafide analogs in toluene followed by precipitation with excess gaseous hydrochloric acid, as described by Zee-Cheng and Cheng (U.S. Pat. No. 4,665,071; 1987). The mitonafide hydrochloride, of unspecified stoichiometry and hydration, obtained by this reaction is a reddish brown precipitate, containing 12% by weight of tar with no differential solubility between water and alcohol, again requiring multiple recrystallizations to afford a hydrochloride salt material of suitable quality for pharmaceutical use.
In the present invention, as shown in FIG. 1, it has now been found that the isolation of the mitonafide moiety (III) from the ethanolic reaction mixture is facilitated by admixture and complete dissolution of a suitable organic carboxylic acid compound, which upon cooling affords a near colorless adduct upon crystallization from a mother liquor which retains the preponderance of colored impurities which might otherwise have co-crystallized as in the case of the prior art. In contrast to the synthetic approach of Brana and associates (Eur. J. Med. Chem 16:207, 1981; U.S. Pat. No. 4,204,063; 1980), described hereinabove, the mitonafide is obtained directly as an organic salt, in the first step of the synthesis for the target amonafide compound, rather than post facto in accordance with the alternate teachings as in U.S. Pat. No. 5,420,137 (1995) relating to the monohydrochloride or monomethylsulfate salts of amonafide obtained by controlled titration of amonafide itself at the end of the reaction sequence.
Those skilled in the art will also recognize that salt formation of mitonafide as an isolation step as taught by U.S. Pat. No. 4,665,071 (1987) cannot be construed as an obvious precedent for the instant invention insofar as organic acids, which are the salt forming reagents in this invention are known to be insoluble in toluene, and similar non-polar solvents, even at reflux. Unlike gaseous HCl used in accordance with the prior art on mitonafide salt isolation, the organic acids of this invention are solids which can be metered with accuracy so as to achieve a precise titration stoichiometry, an elusive objective when the dispensing of gaseous acids is called for as the prevailing alternative.
The novelty of the invention described here, when contrasted to prior art, is further affirmed by the unexpected finding concerning the catalytic hydrogenolysis of the nitro substituents in the mitonafide structural skeleton.
In the specific prior art on the formation of amonafide salts, congeneric with Stucture IV in FIG. 1, Brana and associates (U.S. Pat. No. 5,420,137; 1995) fail to describe the properties of the precursor mitonafide nor do they describe the method of hydrogenation to the resulting amonafide free base. However, in prior disclosures (Spanish Patent 533,542; 1983) on a method for the industrial production specifically of amonafide, these same authors indicate that nitro reduction of the precursor mitonafide free base is effected with 10% palladium-on-carbon (Pd/C) via transfer hydrogenation in the presence of excess hydrazine under refluxing ethanolic conditions. This procedure is also summarized as the preferred approach in the chemical review literature by the same authors (Ars Pharmaceutica 36:377-415, 1995).
Those skilled in the art would recognize that such an approach could not be practiced if the mitonafide precursor were composed of a pre-formed acid salt. Under such circumstances, one might reasonably expect that the hydrazine donor reagent would be neutralized by ion exchange and become unavailable as a substrate for diimide formation, which is the active reducing species catalyzed by Pd/C. In effect, any followers of the teachings of Brana and associates would have contemplated only the use of free-base precursors rather than resorting to the less obvious alternative, namely direct reduction of a mitonafide salt as taught in this patent.
Within the larger scope of organic functional group transformations, those skilled in the art will recognize that the catalytic hydrogenation of aryl nitro compounds to the corresponding substituted anilines is usually practiced in ethanol, mixtures of ethanol and water, or in the so called universal solvents (e.g. dimethyformamide and dimethylacetamide) which are resistant to hydrogenation. Respected, classical monographs on the subject by P. N. Rylander (Catalytic Hydrogenation in Organic Synthesis, New York: Academic Press, 1979) and M. Freifelder (Practical Catalytic Hydrogenation, New York: Wiley, 1971) acknowledge that the solubility of aryl nitro compounds in general precludes use of water as the hydrogenation medium.
These experts also indicate that the preferred source of protons to effect suppression of the imine and oxime by-products of incomplete hydrogenation is achieved by admixture of the substrate with hydrochloric acid. Even in the presence of mineral acids, hydrogenations in water are poorly documented and considered idiosyncratic in both the traditional and current hydrogenation laboratory and industrial practice. Use of organic acids, such as acetic acid or formic acid, has been described, but with the caveat that dehydrative acylation will occur, thus affording the corresponding N-acyl aryl-amines as yield-lowering contaminants.
Thus, in the context of this invention, neither the specific literature on amonafide synthesis nor on methods of hydrogenation can be cited as precedent for the non-obvious chemical manipulations which were found to be advantageous here. First, the use of organic carboxylic acid compounds to effect purification and isolation of mitonafide and its analogs has not been described heretofore. Second, application of organic carboxylic acid salts of mitonafide and its analogs as direct precursors for catalytic hydrogenation has not be considered or promulgated as an effective practice. Third, the high degree of water solubility of these organic salts, itself an unexpected phenomenon, coupled with the reluctance among experts to recommend catalytic hydrogenations in water as the sole solvent, would have precluded exploration of the novel approach presented here.
Beyond these issues which demonstrate non-obviousness, there exist further practical advantages to the use of organic carboxylic acid compounds, and especially their preferred analogs, the organic carboxylic diacid compounds, in the context of this invention. The resulting aralkyl naphthalimide salts show water solubilites as high as 1:1 by proportional admixture in contrast to the mono or divalent salts of hydrochloric, methanesulfonic, or of other mineral acids, whose solubilities fall below 10% by weight. Bulk processing is facilitated for purposes of industrial synthesis, filtration, purification, and dispensing of dosage units prior to sterile filtration and lyophilization.
In dry form, these organic carboxylic acid salts show higher bulk density, porosity and compaction than their analogs mineral acid salts, while presenting lower hygroscopicity. Thus, they are more suitable for processing by direct pressing, rather than solely by granulation or agglomeration.
In terms of biological burden, the organic carboxylate anions present no electrolyte load, unlike the mineral acid anions, and are biodegradable through normal cellular pathways of intermediary metabolism. In either the case of inorganic or organic acid anions, it is a matter of record that these species provide charge balance, solubility, mechanical, adsorptive or absorptive properties to the drug moiety to which they are attached. However, it is known throughout the practice of medicinal and pharmaceutical chemistry that the salt form per se will not affect the pharmacological activity, which in the case of the polycyclic aryl and aralkylamine containing intercalator drugs is their antitumor action.
For example, in reviewing the current pharamacopoeia, the intercalator drug ametantrone is known to be equally active as the free base, hydrochloride, monoacetate and diacetate, the difference in the salt form being their bulk formulation properties. Its analog, NSC-639366, on the other hand is being developed preclinically as the fumarate salt, in preference to the hydrochloride or acetate. In the case of asulacrine, the preferred salt form is the isethionate. For crisnatol and exatecan, it is the mesylate which is pharmaceutically most suitable. Many other drug classes, with diverse modes of action, have been developed as salts of organic acids. For example, the L-malate salts of clebopride, an antinausea medication, of almotriptan, an antimigraine medication, and of pizotifen, an antihistamine, have exhibited enhanced solubility without alteration of their respective medicinal properties.